苦参碱对人横纹肌肉瘤RD细胞MAPK/ERK信号通路的影响

Influence of Matrine on MAPK/ERK signaling pathways of humanrhabdomyosarcoma RD cells

目的探讨苦参碱对人横纹肌肉瘤(RMS)RD细胞ERK通路的影响。方法取对数生长期的细胞,随机分为对照组(含细胞无药物)及3个药物干预组(分别为U0126 10μmol组、苦参碱1.0 mg/m L组和U0126 10μmol与苦参碱1.0 mg/m L共同作用组)。蛋白质印迹法(Western blotting)检测p44/p42MAPK(ERK1/2)及Phospho-p44/p42MAPK(p-ERK1/2)的表达;反转录聚合酶链反应(RT-PCR)方法检测erk m RNA的表达。最后采用SPSS 16.0统计软件进行单因素方差分析。结果 Western blotting法检测ERK1/2、p-ERK1/2的表达,对照组(U0126 10μmol组,苦参碱1.0 mg/m L组及U0126 10μmol与苦参碱1.0 mg/m L共同作用组)的灰度值分别为:2.84±0.03、2.64±0.03、2.14±0.01、2.10±0.02及1.76±0.15、1.37±0.19、1.21±0.24、1.19±0.22。与对照组比较,苦参碱1.0 mg/m L组及苦参碱与U0126共同作用组对ERK1/2、p-ERK1/2蛋白表达量明显降低,差异均有统计学意义(P<0.01);RT-PCR方法检测erk m RNA的表达,对照组(U0126 10μmol组,苦参碱1.0 mg/m L组及U0126 10μmol与苦参碱1.0 mg/m L共同作用组)的灰度比值分别为0.59±0.04、0.57±0.04、0.25±0.02和0.18±0.01,苦参碱1.0 mg/m L组及苦参碱与U0126共同作用组erk m RNA的表达量较对照组明显降低,差异均有统计学意义(P<0.01)。结论苦参碱在体外环境下作用RMS-RD细胞,可抑制erk m RNA的表达及ERK1/2蛋白磷酸化的活性。

Objective To explore the influence of Matrine on MAPK/ERK signaling pathways of human rhabdomyosarcoma（RMS） RD cells. Methods The cells at logarithm growth phase were chosen and randomly divided into the control group（including cells without Matrine）and three intervention groups of different concentrations（respectively for U0126 10 um,Matrine 1.0mg/mL,U0126 10 um combined with Matrine 1.0 mg/mL）. The expressions of p44/p42MAPK（ERK1/2） and Phosopho-p44/p42MARK（p-ERK1/2） were detected by Western blotting,and the expression of erk m RNA was detected by reverse transcriptionpolymerase chain reaction（RT-PCR）. One-way analysis of variance was performed by SPSS16.0 statistical software. Results The grey values of expression of ERK1/2,p-ERK1/2 in the control group,U0126 10 μmol group,Matrine 1.0 mg/mL group and U012610 μmol combined with Matrine 1.0 mg/mL group were（2.84±0.03）,（2.64±0.03）,（2.14±0.01）,（2.10±0.02）;（1.76±0.15）,（1.37±0.19）,（1.21±0.24）,（1.19±0.22） by Western blotting respecitvely,compared with the control group,the Matrine 1.0 mg/mL and Matrine 1.0 mg/mL combined U0126 10 μmol groups ERK1/2,p-ERK1/2 protein expressions were statistically significant difference（P〈0.01）;The expression of erkm RNA in the control group,U0126 10 μmol group,Matrine 1.0 mg/mL group and U0126 10 μmol combined with Matrine 1.0 mg/mL group were（0.59±0.04）,（0.57±0.04）,（0.25±0.02）and（0.18±0.01） by RT-PCR,significant differences were found in the Matrine 1.0 mg/mL group and expression amount of Matrine 1.0 mg/mL combined U012610 μmol compared with the control group（P〈0.01）. Conclusion Matrine may inhibit the RMS-RD cells expression and the phosphorylated activity of ERKm RNA ERK1/2 protein in vitro.